Squalene epoxidase (SE) and oxidosqualene cyclase (OSC) are the two key enzymes required for the production of lanosterol from an acyclic polyene precursor. Recently, these two enzymes, as well as squalene synthetase, have emerged as important new pharmaceutical targets for a dozen companies worldwide. We propose to continue our efforts to determine the molecular interactions involved in substrate binding and catalysis by exploiting the techniques of synthetic chemistry, protein biochemistry, molecular biology, and structural biology. Efficient affinity probes allowed purification and active site labeling of these two enzymes from rat and pig liver. SE from both sources has been affinity purified using a substrate mimic and photoaffinity labeled using a tritiated inhibitor analog: active site sequence analysis and physicochemical characterization have provided several unexpected results. OSC from both sources has been purified, modified with a tritium-labeled, mechanism-based inhibitor, and the active site amino acids sequenced. A novel aromatic-rich repeating motif and an Asp-rich cation binding region were identified. Using amino acids obtained from partially-sequenced peptide fragments, cloning of a cDNA for rat OSC will be attempted using both antibody screening and PCR amplification from a rat liver cDNA library in lambdaZAP. Use of rat cDNAs to complement the OSC-deficient erg7 yeast strain will also be attempted. The rat cDNA will be used to obtain a pig cDNA as well. Second, a similar strategy will be used for rat and pig SE when active site amino acid sequences are obtained. Third, OSC and SE cDNAs will be truncated to allow secretion of soluble forms, if possible, and expression systems will be developed to obtain multimilligram quantities for structural studies. Fourth, expression cassette PCR and site-directed mutagenesis will be employed to determine domains and residues respectively involved in substrate binding and catalysis. Fifth, catalytically-active domains of appropriate size will be crystallized and/or studied in liganded and unliganded states by multidimensional NMR. Sixth, OSC is extensively O-glycosylated. Studies of the native glycoproteins and derived glycopeptides will be undertaken using MALDI-MS- MS, and mutations will be made to determine importance of glycosylation sites. Seventh, effects of pressure on the rate and product distributions of OSC will be undertaken with recombinant enzymes.